Biology subjects

Roversi, P.

Publications and source records attributed to Roversi, P..

8 recordsLinked to original sources

bioRxiv2026

Structural, dynamic, and evolutionary determinants of substrate binding in the tetrameric 6-phosphogluconate dehydrogenase from Gluconobacter oxydans

6-Phosphogluconate dehydrogenases (6PGDHs) catalyze a key oxidative step in the oxidative pentose phosphate pathway (oxPPP), a route essential for NAD(P)H generation and carbon metabolism in bacteria and eukaryotes. While the structural basis of substrate recognition is well established for long-chain dimeric 6PGDHs, the mechanisms used by short-chain tetrameric enzymes remain poorly defined. Here, we present a 2.0 [A] crystal structure of tetrameric 6PGDH from Gluconobacter oxydans (Go6PGDH) in complex with 6-phosphogluconate (6PG) and integrate it with evolutionary, computational, and functional analyses. The structure shows that, unlike dimeric homologs, tetrameric Go6PGDH does not undergo a domain-closure transition upon ligand binding. Instead, 6PG induces a compaction of the tetramer mediated by two conserved C-terminal elements: an inter-protomer ionic "lock" and an intra-subunit C-terminal "latch" that together stabilize a closed catalytic pocket. Molecular-dynamics simulations identify His328 as a central residue that couples C-terminal tail closure to direct ligand coordination, and mutagenesis analysis confirms its essential role in catalytic efficiency. Thermodynamic measurements reveal that 6PG binding is strongly enthalpy-driven, consistent with the formation of an ordered hydrogen-bonding and electrostatic network in the closed conformation. These findings define a substrate-induced quaternary-tightening mechanism unique to tetrameric 6PGDHs and illustrate how a conserved C-terminal module has been adapted across the family to regulate substrate binding and catalysis.

biochemistry↗

bioRxiv2025

Mathematical modelling of mutant glycoprotein fate in the ER

BackgroundThe endoplasmic reticulum (ER) coordinates glycoprotein quality control through competing pathways: ER quality control (ERQC) and ER-associated degradation (ERAD). The fate of a glycoprotein is tied to its N-glycan(s) structure, which serves as a "molecular barcode" for quality control. In particular, modulation of ERQC/ERAD can increase leakage of responsive (i.e., non-inactive) glycoprotein mutants that remain misfolded yet retain biological activity once outside the ER. Rescue of secretion of such mutants is a promising therapeutic strategy for the therapy of congenital rare disease due to a missense mutation in a secreted glycoprotein gene, but the relative merits of UGGT vs. EDEM inhibition to release a mutant from the ER remain to be explored. MethodsAn ordinary differential equation (ODE) framework ("ER Glycoprotein Outcome", ERGO) evaluates time-dependent ER concentrations of 13 N-glycospecies of a single mutant glycoprotein (G1M9, G1M8B, G1M8C, G1M7BC, M9, M8A, M8B, M8C, M7AB, M7AC, M7BC, M6, M5) within the ER lumen, under a set of chosen hypotheses. All reactions follow mass-action kinetics with ER enzymes/lectins kinetic constants and concentrations as fixed parameters. The ODE system is solved in Python with adaptive Runge-Kutta integration. Simulations initialise from a single glycoform (M9, 1 {micro}M) and track glycoform distributions over [~]28 h. Starting glycoform concentrations and overall time can be customised. Given the lack of comprehensive kinetic data in the literature, parameter values reflect educated guesses, rather than measured constants. ResultsFour simulation scenarios explore the dynamics of ERQC vs ERAD: (A) baseline scenario with all enzymes active; (B) UGGT inhibition, which abolishes re-glucosylation, disrupts CNX/CRT cycles, and accelerates substrate flux toward ERAD-prone glycoforms; (C) inhibition of all ER mannosidases, preventing trimming-dependent ERAD commitment and prolonging ER retention of early glycoforms; (D) combined UGGT and ER mannosidases inhibition, mimicking experimental rescue of misfolded substrates by blockade of both glucosylation-driven ER retention and trimming-driven degradation signals. We further quantify the rate of CNX/CRT engagement ( carousel rides) by combining analytical estimates with Monte Carlo-style simulations of individual mutant glycoprotein fates.

bioinformatics↗

bioRxiv2025

Structure and function of the EDEM:PDI ERAD checkpoint complex

The ERAD-L checkpoint complex de-mannosylates misfolded glycoproteins with lumenal defects, targeting them to retrotranslocation, ubiquitination, and proteasomal degradation. Commitment to ERAD-L requires an Endoplasmic Reticulum-Degradation Enhancing alpha-Mannosidase-like protein (EDEM) and its associated Protein Disulfide Isomerase (PDI). We determined Cryo-EM structures of the Chaetomium thermophilum EDEM:PDI heterodimer, both by itself and in complex with a classic ERAD substrate, the alpha1-antitrypsin Null Hong Kong mutant (A1AT-NHK). The EDEM catalytic domain nestles within the PDI arc. One intermolecular disulfide, between the a' domain of PDI and the first conserved cysteine of the EDEM linker (Cys A), stably links the two proteins. A second intermolecular disulfide, between the second conserved cysteine of the EDEM linker (Cys B) and the PDI a domain, stabilises the compact apo conformation. In the substrate-bound complex, the Cys B intermolecular disulfide is reduced and the catalytic CXXC motif of the PDI a domain oxidised. Release of the Cys B linkage increases the conformational freedom of the EDEM linker, allowing the EDEM C-terminal domains to adopt both proximal and distal conformations relative to the catalytic domain. The structures provide a framework for discovery of EDEM:PDI modulators, with potential applications in virology, rare genetic disease and cancer, and as reagents for glycoprotein quality-control engineering.

biochemistry↗

bioRxiv2023

Characterising the Protein-Protein Interaction Between MDM2 and 14-3-3σ; Proof of Concept for Small Molecule Stabilisation

Mouse Double Minute 2 (MDM2) is a key negative regulator of the tumour suppressor protein p53. MDM2 overexpression occurs in many types of cancer and results in the suppression of wild type p53. The 14-3-3 family of adaptor proteins are known to bind to MDM2 and the 14-3-3{sigma} isoform controls MDM2 cellular localisation and stability to inhibit its activity. Therefore, small molecule stabilisation of the 14-3-3{sigma}/MDM2 protein-protein interaction (PPI) is a potential therapeutic strategy for the treatment of cancer. In this work we provide a detailed biophysical and structural characterisation of the phosphorylation-dependent interaction between 14-3-3{sigma} and peptides that mimic the 14-3-3 binding motifs within MDM2. The data show that di-phosphorylation of MDM2 at S166 and S186 is essential for high affinity 14-3-3 binding and that the binary complex formed involves one MDM2 di-phosphorylated peptide bound to a dimer of 14-3-3{sigma}. Each of the two phosphorylated stretches of MDM2 occupies one of the two binding grooves of a 14-3-3{sigma} dimer, a novel model for binding of di-phosphorylated peptides to 14-3-3 proteins. In addition, we show that the 14-3-3{sigma}/MDM2 interaction is amenable to small molecule stabilisation. The natural product fusicoccin A forms a ternary complex with a 14-3-3{sigma} dimer and an MDM2 di-phosphorylated peptide resulting in stablisation of the 14-3-3{sigma}/MDM2 PPI. This work serves as a proof-of-concept of the drugability of the 14-3-3/MDM2 PPI and paves the way toward the development of more selective and efficacious small molecule stabilisers.

biochemistry↗

bioRxiv2023

Rescue of secretion of a rare-disease associated mis-folded mutant glycoprotein in UGGT1 knock-out mammalian cells

Endoplasmic reticulum (ER) retention of mis-folded glycoproteins is mediated by the ER- localised eukaryotic glycoprotein secretion checkpoint, UDP-glucose glycoprotein glucosyl-transferase (UGGT). The enzyme recognises a mis-folded glycoprotein and flags it for ER retention by reglucosylating one of its N-linked glycans. In the background of a congenital mutation in a secreted glycoprotein gene, UGGT-mediated ER retention can cause rare disease even if the mutant glycoprotein retains activity ("responsive mutant"). Here, we investigated the subcellular localisation of the human Trop-2 Q118E variant, which causes gelatinous drop- like corneal dystrophy (GDLD). Compared with the wild type Trop-2, which is correctly localised at the plasma membrane, the Trop-2-Q118E variant is found to be heavily retained in the ER. Using Trop-2-Q118E, we tested UGGT modulation as a rescue-of-secretion therapeutic strategy for congenital rare disease caused by responsive mutations in genes encoding secreted glycoproteins. We investigated secretion of a EYFP-fusion of Trop-2-Q118E by confocal laser scanning microscopy. As a limiting case of UGGT inhibition, mammalian cells harbouring CRISPR/Cas9-mediated inhibition of the UGGT1 and/or UGGT2 gene expressions were used. The membrane localisation of the Trop-2-Q118E-EYFP mutant was successfully rescued in UGGT1-/-and UGGT1/2-/- cells. UGGT1 also efficiently reglucosylated Trop-2-Q118E-EYFP in cellula. The study supports the hypothesis that UGGT1 modulation constitutes a novel therapeutic strategy for the treatment of Trop-2-Q118E associated GDLD, and it encourages the testing of modulators of ER glycoprotein folding Quality Control (ERQC) as broad-spectrum rescue- of-secretion drugs in rare diseases caused by responsive secreted glycoprotein mutants. SynopsisDeletion of the UGGT1 and UGGT1/2 genes in HEK 293T cells rescues secretion of an EYFP-fusion of the human Trop-2-Q118E glycoprotein mutant. The mutant is retained in the secretory pathway in wild type cells and it localises to the cell membrane in UGGT1-/- single and UGGT1/2-/- double knock-out cells. The Trop-2-Q118E glycoprotein disease mutant is efficiently glucosylated by UGGT1 in human cells demonstrating that it is a bona fide cellular UGGT1 substrate. O_FIG O_LINKSMALLFIG WIDTH=120 HEIGHT=200 SRC="FIGDIR/small/542711v1_ufig1.gif" ALT="Figure 1"> View larger version (63K): org.highwire.dtl.DTLVardef@ac29f1org.highwire.dtl.DTLVardef@f5bcbforg.highwire.dtl.DTLVardef@13ab5a1org.highwire.dtl.DTLVardef@16a9406_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2022

Cadmium treatment induces endoplasmic reticulum stress and unfolded protein response in Arabidopsis thaliana

We report about the response of Arabidopsis thaliana to chronic and temporary Cd2+ stress, and the Cd2+ induced activation of ER stress and unfolded protein response (UPR). Cd2+-induced UPR proceeds mainly through the bZIP60 arm, which in turn activates relevant ER stress marker genes such as BiP3, CNX, PDI5 and ERdj3B in a concentration- (chronic stress) or time- (temporary stress) dependent manner. A more severe Cd-stress triggers programmed cell death (PCD) through the activation of the NAC089 transcription factor. Toxic effects of Cd2+ exposure are reduced in the Atbzip28/bzip60 double mutant in terms of primary root length and fresh shoot weight, likely due to reduced UPR and PCD activation. We also hypothesised that the enhanced Cd2+ resistance of the Atbzip28/bzip60 double mutant is due to an increase in brassinosteroids signaling, since the amount of the brassinosteroid insensitive1 receptor (BRI1) protein decreases under Cd2+ stress only in Wt plants. These data highlight the complexity of the UPR pathway, since the ER stress response is strictly related to the type of the treatment applied and the multifaceted connections of ER signaling. The reduced sensing of Cd2+ stress in plants with UPR defects can be used as a novel strategy for phytoremediation.

plant biology↗

bioRxiv2022

A quinolin-8-ol sub-millimolar inhibitor of UGGT, the ER glycoprotein folding quality control checkpoint

The Endoplasmic Reticulum (ER) glycoprotein folding Quality Control (ERQC) machinery aids folding of glycoproteins in the ER. Misfolded glycoprotein recognition and ER-retention is mediated by the ERQC checkpoint enzyme, the 170 kDa UDP-Glucose glycoprotein glucosyltransferase (UGGT). UGGT modulation is a promising strategy for broad-spectrum antivirals, rescue-of-secretion therapy in rare disease caused by responsive mutations in glycoprotein genes, and many cancers, but to date no selective UGGT inhibitors are known. Towards the generation of selective UGGT inhibitors, we determined the crystal structures of the catalytic domain of Chaetomium thermophilum UGGT (CtUGGTGT24), alone and in complex with the inhibitor UDP-2-deoxy-2-fluoro-D-glucose (U2F). Using the CtUGGTGT24 crystals, we carried out a fragment-based lead discovery screen via X-ray crystallography and discovered that the small molecule 5-[(morpholin-4-yl)methyl]quinolin-8-ol (5M-8OH-Q) binds a CtUGGTGT24 WY conserved surface motif that is not present in other GT24 family glycosyltransferases. The 5M-8OH-Q molecule has a 613 {micro}M binding affinity for human UGGT1in vitro as measured by saturation transfer difference NMR spectroscopy. The 5M-8OH-Q molecule inhibits both human UGGT1and UGGT2 activity at concentrations higher than 750 {micro}M in modified HEK293-6E cells. The compound is toxic in cellula and in planta at concentrations higher than 1 mM. A few off-target effects are also observed upon 5M-8OH-Q treatment. Based on an in silico model of the interaction between UGGT and its substrate N -glycan, the 5M-8OH-Q molecule likely works as a competitive inhibitor, binding to the site of recognition of the first GlcNAc residue of the substrate N -glycan. Significance StatementWhen a candidate drug target is the product of a housekeeping gene - i.e. it is important for the normal functioning of the healthy cell - availability of inhibitors for tests and assays is of paramount importance. One such housekeeping protein is UGGT, the enzyme that makes sure that only correctly folded glycoproteins can leave the endoplasmic reticulum for further trafficking through the secretory pathway. UGGT is a potential drug target against viruses, in certain instances of congenital rare disease, and against some cancers, but no UGGT inhibitors are known yet. We discovered and describe here a small molecule that binds human UGGT1 in vitro and inhibits both isoforms of human UGGT in cellula. The compound paves the way to testing of UGGT inhibition as a potential pharmacological strategy in a number of medical contexts.

biochemistry↗

bioRxiv2019

Clamping, bending, and twisting inter-domain motions in the misfold-recognising portion of UDP glucose:glycoprotein glucosyl-transferase.

UDP-glucose:glycoprotein glucosyltransferase (UGGT) is the only known glycoprotein folding quality control checkpoint in the eukaryotic glycoprotein secretory pathway. When the enzyme detects a misfolded glycoprotein in the Endoplasmic Reticulum (ER), it dispatches it for ER retention by re-glucosylating it on one of its N-linked glycans. Recent crystal structures of a fungal UGGT have suggested the enzyme is conformationally mobile. Here, a negative stain electron microscopy reconstruction of UGGT in complex with a monoclonal antibody confirms that the misfold-sensing N-terminal portion of UGGT and its C-terminal catalytic domain are tightly associated. Molecular Dynamics (MD) simulations capture UGGT in so far unobserved conformational states, giving new insights into the molecules flexibility. Principal component analysis of the MD trajectories affords a description of UGGTs overall inter-domain motions, highlighting three types of inter-domain movements: bending, twisting and clamping. These inter-domain motions modify the accessible surface area of the enzymes central saddle, likely enabling the protein to recognize and re-glucosylate substrates of different sizes and shapes, and/or re-glucosylate N-linked glycans situated at variable distances from the site of misfold. We propose to name "Parodi limit" the maximum distance between a site of misfolding on a UGGT glycoprotein substrate and an N-linked glycan that monomeric UGGT can re-glucosylate on the same glycoprotein. MD simulations estimate the Parodi limit to be around 60-70 [A]. Re-glucosylation assays using UGGT deletion mutants suggest that the TRXL2 domain is necessary for activity against urea-misfolded bovine thyroglobulin. Taken together, our findings support a "one-size-fits-all adjustable spanner" substrate recognition model, with a crucial role for the TRXL2 domain in the recruitment of misfolded substrates to the enzymes active site.

biochemistry↗